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J Biol Chem. 2010 Jan 29;285(5):3310-8. doi: 10.1074/jbc.M109.060418. Epub 2009 Nov 30.

Aminopeptidase fingerprints, an integrated approach for identification of good substrates and optimal inhibitors.

Author information

1
Apoptosis and Cell Death Research Program, the Burnham Institute for Medical Research, La Jolla, California 92037, USA. mdrag@burnham.org

Abstract

Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.

PMID:
19948737
PMCID:
PMC2823418
DOI:
10.1074/jbc.M109.060418
[Indexed for MEDLINE]
Free PMC Article

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